The Effect And The Mechanism Of HPV-16E7 On DSB Repair Through SIRT7

BackgroundCervical cancer is one of the most common gynecological tumors which poses a serious threat to women's health.Chemotherapy is an important treatment for cervical cancer.Some chemotherapy regents induce DNA damage to kill tumor cells.DNA double-strand break(DSB)is one of the most serious DNA damage.The repair of DSB damage by tumor cells can enhance chemotherapy resistance,resulting in poor prognosis.Persistent infection of high-risk human papillomavirus(HPV)is a major cause for cervical cancer,with HPV-16 being the most common type.HPVs encode 6 early proteins and 2 late proteins.E6 and E7 are the main oncoproteins,which are involved in regulating a variety of biological activities related to malignant transformation of cervical epithelial cells,such as cell proliferation,apoptosis,migration and invasion,and maintenance of genomic stability.The research on DNA repair regulated by E7 is still controversial.It is reported that HPV-16E7 up-regulates the expression of DNA repair proteins,such as ATM,Chk2,etc.,thereby promoting DNA damage repair.It is also reported that HPV-16E7 prolongs the presence of yH2AX foci and inhibits DSB repair.The mechanism of HPV-16E7 regulating DSB is still unclear.Sirtuin7(SIRT7),a member of Sirtuins,is a NAD(+)dependent histone deacetylase,which plays a significant role in many physiological and pathological processes including DNA repair and maintenance of genetic stability.Previous reports and our preliminary studies have shown that HPV-16E7 up-regulates the expression of SIRT7.It is still unknown whether HPV-16E7 regulates DSB repair through SIRT7.MRN(Mrell-Rad50-Nbsl)complex is important for DNA replication,cell cycle,maintenance of telomere and genomic stability.It can recognize DNA damage,recruit and activate DSB repair proteins such as ATM.It is reported that SIRT1 deacetylates Nbs1 to promote the formation of MRN complex.The information gathered from BioGRID database shows that SIRT7 interacts with Mre11.The mechanism of SIRT7 regulating Mre11 activation and MRN complex formation remains to be further elucidated.ObjectiveTo study the role and molecular mechanism of SIRT7 in HPV-16E7 regulated DSB repair.Methods and result1.HPV-16E7 promotes DSB repair at low damage level while inhibits DSB repair at high damage level.Western Blot results showed that comparing with control cells(pBabe-puro),HaCat cells infected with retrovirus encoding HPV-16E7(pBabe-HPV-16E7)had a higher expression of HPV-16E7 and p53.The results of flow cytometry and neutral comet assays showed that after being treated with 10?g/ml bleomycin for 0.5h and releasing for 1h,The amount of yH2AX foci was less and olive tail moment(OTM)was lower in experimental group,while after being treated with 20?g/ml bleomycin,there were more ?H2AX foci and higher OTM in experimental group.The results of Western Blot detecting DNA repair proteins showed that HPV-16E7 up-regulated the expression of pBRCA1(a homologous recombination related protein)and XRCC4(a non-homologous end joining related protein)at low-level DSB damage while down-regulated the expression of these repair proteins at high-level DSB damage.2.HPV-16E7 regulates DSB repair through SIRT7.Quantitative real-time polymerase chain reaction(QRT-PCR)and Western Blot results showed that HPV-16E7 increased SIRT7 expression at low-level DSB damage while down-regulated SIRT7 expression at high-level DSB damage.We transfected HPV-16E7 overexpressing HaCat cells and control cells with siRNA-SIRT7 and then treated the cells with different concentrations of bleomycin for 0.5h and released for 1h.Flow cytometry and neutral comet assay results showed that comparing with control group,the experimental group had more ?H2AX foci and higher OTM,suggesting that SIRT7 knock-down can partly reverse the impact of HPV-16E7 on DSB repair.Western Blot results showed that siRNA-SIRT7 mediated SIRT7 knock-down up-regulated pBRCA1 while down-regulated XRCC4.3.Up-regulation of SIRT7 promotes DSB repair while down-regulation of SIRT7 inhibits DSB repair.We transfected pcDNA3.1-Flag-SIRT7 into HaCat cells and treated the cells with different concentrations of bleomycin for 0.5h and released 1h.Then the cells were collected and detected by yH2AX flow cytometry and neutral comet assays,and the results suggested that SIRT7 overexpression promoted DSB repair.Western Blot results showed that SIRT7 overexpression increased XRCC4 expression while decreased pBRCA1 expression.Western Blot results showed that lentivirus encoding shRNA-SIRT7 can effectively decreased the expression of SIRT7 in HaCat cells.yH2AX flow cytometry and neutral comet assays results suggested that the DSB damage level of the shRNA-SIRT7 group was higher than that of the control group.Detection of DNA repair proteins revealed that SIRT7 knock-down up-regulated the expression of pBRCA1 while down-regulated the expression of XRCC4.4.The restoration of SIRT7(wt)promotes DSB repair.After restoration of wide-type SIRT7(wt)and SIRT7(H187Y)mutant which does not have deacetylation function,the SIRT7 low-expressing cells were treated with different concentrations of bleomycin for 0.5h and released for 1h.?H2AX flow cytometry and neutral comet assays were performed to detect the DSB damage levels of cells and the results suggested that restoration of SIRT7(wt)promoted DSB repair but SIRT7(H187Y)did not have such effect.Western Blot results showed that SIRT7(wt)promoted the expression of XRCC4 while inhibited the expression of pBRCA1 but SIRT7(H187Y)did not have such effect.5.SIRT7 deacetylates Mrell,thereby promoting Mre11 phosphorylation and MRN formation.Co-immunoprecipitation(Co-IP)results suggested that SIRT7 co-precipitated with Mrell and Mrell also co-precipitated with SIRT7.Immunoprecipitation(IP)results suggested that shRNA-SIRT7 mediated SIRT7 knock-down increased Mre11 acetylation,thereby inhibiting the phosphorylation of Mre11 at Ser676.Restoration of SIRT7(wt)partly reversed the impact of SIRT7 knock-down on Mre11 acetylation and phosphorylation,while SIRT7(H187Y)did not have such impact.The results of immunofluorescence and Co-IP showed that,SIRT7 knock-down inhibited the co-localization and co-precipitation of the three components of MRN complex.Restoration of SIRT7(wt)partly reversed the impact of SIRT7 knock-down on MRN complex formation while restoration of SIRT7(H187Y)did not have such function.Conclusions1.HPV-16E7 promotes DNA repair at low-level DSB damage,while inhibits DNA repair at high-level DSB damage.2.HPV-16E7 regulates DSB damage repair through SIRT7.E7 up-regulates SIRT7 expression at low-level DSB damage while down-regulates SIRT7 expression at high-level DSB damage.3.SIRT7 deacetylates Mrell,thereby promoting Mrell phosphorylation at Ser676 and MRN complex formation,and promoting DSB repair.